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Molecular DNA–Based Blood Group Typing
DNA-based testing for blood group antigens has become commonplace in number of clinical situations. These include typing for minor antigens in multiple transfused immunized patients to determine risk for production of additional blood group antibodies, patients with positive direct antiglobulin test (DAT) and serum autoantibody, patients facing chronic transfusion therapy, and for locating antigen-negative blood when no serologic reagent is available, as well as in prenatal medicine to assess risk for hemolytic disease of the newborn (HDFN) and to guide Rh immune globulin (RhIg) therapy for pregnant women.
Determination of blood group antigens by DNA methods (genotyping) is an indirect method for predicting individual’s blood group phenotype, in contrast to direct testing by serologic methods using specific antibody (phenotyping). DNA typing results are often reported as predicted type to distinguish results from testing done by serologic methods.
Most blood group antigens result from single-nucleotide gene polymorphisms (SNPs) inherited in a straightforward Mendelian manner, making assay design and interpretation fairly straightforward. However, ABO and Rh blood groups are more complex. There are >200 different alleles encoding glycosyltransferases responsible for ABO type, and single point mutation in A or B allele can result in inactive transferase, i.e., group O phenotype. Next-generation sequencing (NGS) technology holds promise for routine ABO typing by DNA methods. For Rh system, testing for common antigens D, C/c, and E/e is fairly straightforward in most individuals, but antigen expression is more complex in diverse ethnic groups. There are >200 RHD alleles encoding weak D or partial D phenotypes, and >100 RHCE alleles encoding weak, altered, or novel hybrid Rh proteins. RH genotyping, particularly in minorities, requires sampling of multiple regions of the gene(s) and algorithms for interpretation.
The most commonly used methods for determination of RBC human erythrocyte antigens (HEAs) and human platelet antigens (HPAs) are semiautomated polymerase chain reaction (PCR) using florescent probes with automated readout. Automated methods increase number of target alleles in PCR, allowing determination of numerous antigens in single assay. Most platforms currently available are based on fluorescent bead technology.
Real-Time Polymerase Chain Reaction
Real-time PCR allows automated detection of amplification products and does not require handling of PCR reaction products postamplification. The method is also quantitative, which allows determination of gene copy number. The most common design uses sequence-specific fluorescent probe (TaqMan) that binds to target SNP of interest. The probe has reporter dye (fluorophore) attached to 5′ end and quencher at 3′ end that prevents reporter dye from fluorescing. As target locus is amplified, DNA polymerase encounters bound TaqMan probe and degrades it, allowing reporter to fluoresce when freed from proximal 3′ quencher. Fluorescence amount is directly proportional to release of reporter and PCR product amount.
Bead Technology
This method use multiplexing (amplification of multiple target loci in one assay) with automated detection and interpretation. Many use 96-well format multiplex design that allows for typing multiple samples for many different antigens. Allele-specific capture probes are affixed to beads of many fluorescent colors (>100). Multiple beads are used, with each color targeting different SNPs. Amplified DNA fragments are allowed to anneal to allele-specific capture probes and are then elongated using fluorescent-labeled nucleotides. Beads and associated signals are analyzed by flow cytometer or fluorescence microscopy.
Testing platforms that use bead, or another, DNA array technology, can readily sample multiple genes, and/or regions of genes, and apply automated multifaceted algorithms for accurate interpretation of alleles.
Discrepancies Between Serology (Phenotype) and DNA (Genotype)
When differences between serologic and DNA testing occur, it is important to investigate. This can indicate the presence of novel allele or genetic variant, particularly when testing individuals from diverse ethnic groups. Primary cause of discrepancies between serologic phenotype and DNA genotype when testing donors by large-scale DNA typing has been traced to manual recording errors. Other common causes of discrepancies include the presence of variant alleles encoding weak antigen expression. One example is FYX allele, which encodes amino acid change causing a Fy(b+ w ) weak phenotype. RBCs type as Fy(b−) with most serologic reagents. Prevalence of FYX encoding Fy(b+ w ) in Caucasians is nearly 2%.
DNA testing interrogates single or few SNPs associated with antigen expression and cannot sample every nucleotide in the gene. Consequently, discrepancies will occur when typing patients who have rare or novel silencing mutations that cause loss of antigen expression, resulting in false-positive-predicted antigen type. Silencing mutations can be familial, or common, in particular ethnic group. For example, silencing mutations responsible for S-s-U- phenotypes are common in African black ethnic groups. Fy(a−b−) phenotype found in African blacks is caused by mutation in promoter region of FYB , which disrupts binding site for erythroid transcription factor GATA-1 and results in loss of Duffy expression on RBCs. For accuracy, GATA-1 mutation must be included when typing for Duffy in African blacks. Expression of the protein on endothelium is not altered and Fy(a−b−) individuals with GATA-1 mutations are not at risk for anti-Fy b . Silencing mutations associated with loss of Kidd antigen expression occur more often in Asians, while nucleotide changes encoding amino acid changes that weaken Kidd expression are seen in blacks.
For resolution of discrepancies and to identify new alleles, specialty referral laboratories use methods similar to those used for high-resolution HLA typing, i.e., gene-specific amplification of coding exons followed by sequencing, or alternatively, gene-specific cDNA amplification and sequencing.
Exon-Specific Gene Sequencing
Primers specific for the exon encoding allelic polymorphism, or for each exon of the gene when performing full gene sequencing, are used for amplification. Exon-specific products can be visualized by gel electrophoresis, separated from excess primers, and sequenced by standard Sanger sequencing methods. Computer software programs are used to compare nucleotide sequence obtained to known reference sequence to detect nucleotide changes. Translation programs are used to determine if any nucleotide changes discovered encode amino acid changes in the protein, or alternatively, are synonymous and predicted to be silent.
Gene Sequencing by cDNA Analysis
When gene sequencing is required, usually when investigating the presence of new or novel alleles, sequencing of cDNA synthesized from mRNA is the preferred approach. This requires isolation of mRNA from RBCs. It is important to note that commercial kits available for RNA isolation from blood samples isolate RNA from WBCs, discarding RBC lysate. At least, 0.5 mL of RBCs is needed, as most of the residual mRNA is present in reticulocytes, which represent the minority of cells in nonanemic patient samples. This approach offers advantage that noncoding introns are removed (spliced) and coding region sequence can be analyzed directly. cDNA is synthesized from mRNA using reverse transcriptase and a 3′ gene-specific primer, followed by PCR amplification with 5′ and 3′ gene-specific primers, purification of products, and sequencing.
Applications of DNA-Based Molecular Testing
Transfusion Service Applications
Type Patients Who Have Been Recently Transfused
In patients receiving chronic or massive transfusion, presence of donor RBCs often makes RBC typing by serologic agglutination inaccurate. DNA typing overcomes this limitation and avoids time-consuming and cumbersome cell separation methods to isolate and type the patient’s reticulocytes. DNA assays for blood groups avoid interference from donor-derived DNA by targeting and amplifying a region of the gene common to all alleles. This allows reliable blood group determination with DNA prepared from blood samples collected after transfusion. In transfusion-dependent patients who produce alloantibodies, extended antigen profile is important to determine additional blood group antigens to which the patient can become sensitized.
Type RBCs Coated With Immunoglobulin (Positive DAT)
In patients with RBCs coated with immunoglobulin (IgG), with or without autoimmune hemolytic anemia, presence of bound IgG often makes RBC typing by serologic methods invalid. IgG removal techniques are not always effective and can destroy or weaken antigen of interest. For patients with serum autoantibody, DNA testing allows determination of extended antigen profile to select antigen-negative RBCs for transfusion. This avoids use of “least incompatible” blood for transfusion and allows transfusion of units “antigen-matched for clinically significant blood group antigens” to prevent delayed transfusion reactions and circumvent additional alloimmunization. Importantly, this approach can improve patient care and testing turnaround time by eliminating need for repeat adsorptions to remove autoantibody to rule out new underlying RBC alloantibodies.
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